Figure 9: Role of Rbfox1-iso1 in the dendritic spine morphology in primary cultured hippocampal neurons.

(a) Knockdown of endogenous Rbfox1-iso1. Neurons were transfected with pβAct-EGFP together with pSuper-H1.shLuc (Control) or pSuper-mRbfox1-iso1#1 when isolated, and then fixed at 21 div. Cells were stained with anti-GFP (green) and anti-Rbfox1 (magenta). Double-positive cells for GFP and Rbfox1 were indicated by arrowheads. Quantification of Rbfox1 expression was performed as in Fig. 2e. Scale bar, 20 μm. (b) Effects of Rbfox1-iso1-knockdown on the density and morphology of dendritic spines. Transfection was done as in (a) and stained for GFP. Rescue experiments were done with pCAG-Myc-mRbfox1-iso1R. Magnified images of the indicated areas are also shown. Scale bars, 10 μm (upper panels) and 5 μm (lower panels). (c) Quantitative analyses of density of dendritic spines for each condition in (b). Error bars show SD of the results from 25–30 neurons. Experiments were repeated 3 times with similar results and representative data are shown. **p < 0.01 by Tukey-Kramer LSD. (d) Typical examples of mushroom, stubby, thin filopodia-like and branched spines. (e) Relative abundance of the spine types in neurons transfected as in (b). Relative percentages of each spine type were indicated. Statistical analyses were performed as in (c). **p < 0.01, *p < 0.05 by Tukey-Kramer LSD.